Rapid, reliable, and inexpensive characterization of polymers, particularly the sequencing of nucleic acids, has become increasingly important. One potential application of polymer characterization is in the field of personalized medicine. For example, potential benefits of polymer characterization may include treatment of disease by identifying patients who will gain the greatest benefit from a particular medicine, and those who are most at risk of adverse reactions. The ability to read individual genomes quickly and economically would be a beneficial tool in the development of personalized medicine.
Existing approaches have attempted to address the need for rapid, reliable and inexpensive polymer characterization. For example, some existing approaches use sequencing by synthesis, which includes detection of optical signals during synthesis of complementary deoxyribonucleic acid (DNA) strands. However, sequencing by synthesis produces problems such as, for example, slow reagent cycling times (tens of seconds), short read lengths (tens to hundreds of bases) and expensive reagents. Slow reagent cycling times is a fundamental problem because it results in a need to change chemistry in a flow cell to remove fluorophore from each incorporated base.
Also, other exiting approaches use nanopore sequencing, which includes driving DNA through a nanopore and measuring the electrical current in the DNA as a function of the nucleotides inside the nanopore. Some existing approaches attempt to thread a long DNA molecule through a few nanometer-wide nanopore and use physical differences between the four base types to read the sequence of bases in DNA The price of nanopore sequencing is expected to be very low since the method needs neither expensive chemical reagents nor expensive optical readout. However, single nucleotide resolution has not yet been achieved. Existing approaches using nanopore sequencing cannot resolve a single base, but, rather, require at least a few dozen bases.
Some existing approaches rely on using a readily available-in-nature biological nanopore, that is, α-hemolysin channel (for example, U.S. Pat. No. 5,795,782 entitled “Characterization of individual polymer molecules based on monomer-interface interactions.”). Some existing approaches detect events of DNA translocation through a nanopore by measuring sub-millisecond blockades of ionic current through the nanopore but fail to resolve single bases within the translocated molecule. Despite the ease of obtaining biological nanopores, the difficulties of dealing with unreliable and poorly understood membrane proteins lead many researches to use solid-state nanopores.
Other existing approaches use nanopores with diameters of between two and three nanometers (nm), fabricated by using such materials as Si3N4 or SiO2 (for example, U.S. Pat. No. 6,627,067 entitled “Molecular and atomic scale evaluation of biopolymers,” and U.S. Patent Application No. 2006/0063171 entitled “Methods and apparatus for characterizing polynucleotides.”). Solid-state nanopores also provide the possibility of placing metal electrodes in the vicinity of probed DNA. This arrangement, in theory, allows researchers to measure the tunnel current through a single base, and, consequentially, potentially discriminate the bases of different types. In existing approaches, however, repetitive measurements of tunnel current are necessary to provide enough statistics to determine the base type with a high degree of accuracy.
Existing approaches in the area of mechanical polymer characterization include U.S. Patent Application No. 2006/0057585 entitled “Nanostepper/Sensor Systems and Methods of Use Thereof,” filed Sep. 10, 2004. This approach includes a nanopore system and a first nanostepper system, wherein the nanopore system includes a structure having a nanopore aperture, and the first nanostepper system includes an x-/y-direction moving structure and a first nanostepper arm positioned adjacent the structure.
At present, nanopore sequencing is still theoretical, as single nucleotide resolution has not yet been achieved. One of the possible reasons for such unsuccessful experimental results in existing approaches is that the translocation of DNA through the nanopore is too fast and erratic for current measurement methods to reliably resolve the type of a single nucleotide. Despite attempts to slow down the translocation speed by optimization of various parameters (for example, electrolyte temperature, salt concentration, viscosity, and the electrical bias voltage across the nanopore), existing approaches have still been unsuccessful in attaining single nucleotide resolution.
It would thus be desirable to overcome these and other limitations in existing polymer characterization approaches.